Biochemistry 1996, 35, 9385-9391
9385
Mercaptide Formed between the Residue Cys70 and Hg2+ or Co2+ Behaves as a Functional Positively Charged Side Chain Operative in the Arg70 f Cys Mutant of the Metal-Tetracycline/H+ Antiporter of Escherichia coli† Yuichi Someya and Akihito Yamaguchi* Department of Cell Membrane Biology, Institute of Scientific and Industrial Research, Osaka UniVersity, Ibaraki, Osaka 567, Japan ReceiVed April 30, 1996X
The bacterial tetracycline/H+ antiporter (TetA) mediates active efflux of a chelation complex of tetracycline with a divalent cation such as Mg2+, Co2+, or Mn2+ [Yamaguchi, A., Udagawa, T., & Sawai, T. (1990a) J. Biol. Chem. 265, 4809-4813]. The positive charge of Arg70 in the antiporter is important for the transport function [Yamaguchi, A., Someya, Y., & Sawai, T. (1992c) J. Biol. Chem. 267, 19155-19162]. Out of six site-directed mutants of Arg70, only the Lys70 mutant retained moderate transport activity, whereas the Ser70, Ala70, Trp70, Leu70, and Asp70 mutants had no or extremely low transport activity. In this study, we constructed the Cys70 mutant and found that the Cys70 mutant showed, unexpectedly, a significant activity comparable to that of the Lys70 mutant in the presence of Co2+ ions, whereas it showed very low activity as well as the Ala70 mutant in the presence of Mg2+ or Mn2+ ions. Hg2+, which is known to be a cysteine specific modifier but has no ability to form a complex with tetracycline, caused a dramatic increase in the Vmax value of Co2+-dependent tetracycline transport mediated by the Cys70 mutant without affecting the Km value, whereas activities of the wild-type and the Lys70 and Ala70 mutants were not affected by Hg2+. Hg2+ alone without Co2+ could not support the transport activity at all, because Hg2+ does not form a chelation complex with tetracycline. These observations suggest that a mercaptide formed between the SH group of Cys70 and Hg2+ or Co2+ works as a positively charged side chain like that of Arg or Lys. When the SH group of the Cys70 mutant was masked with modification by sulfhydryl reagents, the residual activity was no longer affected by Hg2+. Inversely, when the Cys70 mutant was preincubated with Hg2+, it was protected from the inactivation by sulfhydryl reagents. These observations also confirm the mercaptide formation between the Cys70 and a divalent cation as a functional side chain. ABSTRACT:
The metal-tetracycline/H+ antiporter [TetA(B)] encoded by transposon Tn10 (McMurry et al., 1980; Yamaguchi et al., 1990a) mediates high-level resistance to tetracyclines (Mendez et al., 1990). This is composed of 401 amino acid residues (Hillen & Schollmeier, 1983; Nguyen et al., 1983), which was estimated to have 12 membrane-spanning R-helices (Eckert & Beck, 1989; Allard & Bertrand, 1992), and belongs to a major facilitator superfamily (Marger & Saier, 1993). Extensive site-directed mutagenesis studies on this protein have been performed (Yamaguchi et al., 1990b, 1991, 1992a-d, 1993a-c; McMurry et al., 1992), and now this protein is a paradigm not only of bacterial drug exporters (Neyfakh et al., 1991; Levy, 1992) but also of antiporters and symporters. It is well-known that transporters belonging to the major facilitator superfamily have a unique conserved sequence motif on their putative cytoplasmic loop region (loop2-3) between transmembrane helices 2 and 3 (Maiden et al., 1987; Henderson, 1990), which is represented as GXXXDRXGRR. The precise role of this motif still remains unknown. We first constructed a complete set of site-directed mutants of † This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education of Japan. Y.S. is a Research Fellow of the Japan Society for the Promotion of Science. * To whom correspondence should be addressed. Telephone: 81-6879-8545. Fax: 81-6-879-8549. E-mail:
[email protected]. X Abstract published in AdVance ACS Abstracts, July 1, 1996.
S0006-2960(96)01026-4 CCC: $12.00
all 10 amino acid residues composing the loop2-3 of TetA (Yamaguchi et al., 1992c). As a result, it was revealed that only 4 residues, Gly62, Asp66, Gly69, and Arg70, out of 10 residues were important for the function or the maintenance of the protein structure. In spite of high conservation among the major facilitator superfamily, mutations of Arg67 and Arg71 caused no significant decrease in activity (Yamaguchi et al., 1992c). As for Asp66, the negative charge is essential for function because only the Glu66 mutant retained very low but significant transport activity (Yamaguchi et al., 1990b). The loop2-3 of TetA contains four positively charged residues and only one negatively charged residue. Among four positively charged residues, the importance of Arg70 was strikingly conspicuous. When Arg70 was replaced with Ser, Ala, Trp, Leu, or Asp, the transport activity was almost lost. Only the Lys70 mutant retained the transport activity, which was about 30% of the wild-type activity (Yamaguchi et al., 1992c). Thus, it seemed that a positive charge at this position was essential. The example other than TetA for site-directed mutagenesis studies of the loop2-3 region of membrane transporters is the R-ketoglutarate permease (KgtP). Seol and Shatkin (1992) confirmed our findings as to the importance of the fifth acidic residue and the ninth basic residue in this motif by constructing corresponding site-directed mutants of KgtP. Very recently, Jessen-Marshall et al. (1995) performed the © 1996 American Chemical Society
9386 Biochemistry, Vol. 35, No. 29, 1996 site-directed mutagenesis study for the loop2-3 region of the lactose permease, which also confirmed the importance of this region although none of the basic residues was essential for lactose transport. The Cys70 mutant of the TetA protein was constructed at first in order to determine whether the side chain is exposed to the medium on the basis of the reactivity of the SH group with N-ethylmaleimide. As a result, the SH group of the Cys70 mutant showed high reactivity to N-ethylmaleimide (manuscript in preparation), confirming that position 70 is located on the surface of the membrane, exposed to the medium. In the course of this experiment, we noticed that the Cys70 mutant had unexpectedly significant tetracycline transport activity in spite of the side chain having no positive charge. In this study, we report the reason why the Cys70 mutant of TetA shows such significant transport activity. EXPERIMENTAL PROCEDURES Materials. [7-3H]Tetracycline was purchased from Du Pont-New England Nuclear. [R-32P]dCTP was purchased from Amersham. All other chemicals were of reagent grade and from commercial sources. Bacterial Strains and Plasmids. Escherichia coli TG1 (Taylor et al., 1985) and W3104 (Yamamoto et al., 1981) were used for plasmid isolation and preparation of inverted membrane vesicles, respectively. pER (Yamaguchi et al., 1992a) is a subclone of the tetA gene used for mutagenesis. Low-copy number plasmid pLGT2 (Yamaguchi et al., 1992a), which was derived from pLG339 (Stoker et al., 1982), contains the entire tetR and tetA genes. Site-Directed Mutagenesis. Mutagenesis for the Arg70 f Cys (R70C) substitution was performed using the Amersham kit (oligonucleotide-directed in vitro mutagenesis system, Ver. 2.1) (Taylor et al., 1985). The nucleotide sequence of the mutagenic primer was 5′-GATTTGGTTGCAGGCCTGTGCTGT-3′ (the codon for Cys70 is underlined), and single-stranded DNA of pER was used as a template. The mutation was detected by the appearance of the StuI site and confirmed by DNA sequencing (Sanger et al., 1977) using an Amersham kit (Sequenase version 2.0). The EcoRVEcoRI fragment containing the R70C mutation was cut out from the mutant pER plasmid and exchanged with the corresponding region of pLGT2 to construct pLGR70C. Preparation of InVerted Membrane Vesicles. Inverted membrane vesicles were prepared by passing E. coli W3104 cells harboring plasmids carrying the wild-type or mutant tetA gene through a French pressure cell, suspended in 50 mM MOPS-KOH1 buffer (pH 7.0) containing 0.1 M KCl, and stored at -80 °C (Yamaguchi et al., 1992c). Transport Assay. [3H]Tetracycline uptake by inverted vesicles was assayed as described previously (Yamaguchi et al., 1992c). Ten microliters of inverted vesicles (3.5 mg of protein/mL) was energized by adding 0.5 µL of 250 mM β-NADH (for background uptake, this step was omitted) at 30 °C. After 1 min, 40 µL of 50 mM MOPS-KOH (pH 7.0), 0.1 M KCl containing 12.5 µM [3H]tetracycline, and 62.5 µM CoCl2 (which gave final concentrations of 10 and 50 µM, respectively) were added, incubated at 30 °C for the indicated times, and filtrated through a Millipore filter (0.45 µm). The filter radioactivities were counted with a 1 Abbreviations: NEM, N-ethylmaleimide; MMTS, methyl methanethiosulfonate; MOPS, 3-(N-morpholino)propanesulfonic acid.
Someya and Yamaguchi liquid scintillation counter. In some assays, 12.5 µM CoCl2 was substituted with 1.25 mM CoCl2, 12.5 µM HgCl2, or a mixture of 62.5 µM CoCl2 and 12.5 µM HgCl2. The kinetic constants for tetracycline transport were measured in the presence of 1 mM CoCl2 and various concentrations (5100 µM) of [3H]tetracycline. H+-Translocation in InVerted Membrane Vesicles. H+ translocation across membranes was measured by monitoring the change in fluorescence of acridine orange (excitation wavelength, 490 nm; emission wavelength, 530 nm) or quinacrine (excitation wavelength, 440 nm; emission wavelength, 500 nm). Typically, 20 µL of inverted vesicles (2 mg of protein/mL) was diluted with 1 mL of 50 mM MOPSKOH (pH 7.0) containing 0.1 M KCl, 10 mM MgSO4, and 1.25 µM acridine orange or 0.8 µM quinacrine. Membranes were energized with a final concentration of 625 µM NADH. At the end of one experiment, NH4Cl was added to a final concentration of 2.5 mM. Other Methods. Measurement of the tetracycline resistance level and Western blotting were performed as described previously (Yamaguchi et al., 1990b, 1992c). RESULTS Construction of the Arg70 f Cys Mutant of the TetA Protein. The Cys70 mutant of TetA was constructed by sitedirected mutagenesis described in Experimental Procedures. E. coli cells carrying a low-copy number plasmid, pLGR70C, encoding the Cys70 mutant TetA showed a moderate level of tetracycline resistance (minimum inhibitory concentration, 12.5 µg/mL), which was about half of the resistance level of the Lys70 mutant (25 µg/mL). The expression of the mutant tetA gene was detected by Western blotting using anti-TetA-C-terminal-peptide antiserum. There was no significant difference in the amounts of the mutant and wildtype TetA proteins (data not shown). Tetracycline Transport ActiVity of InVerted Membrane Vesicles. Figure 1A shows the tetracycline uptake by inverted membrane vesicles containing the mutant or wildtype TetA proteins under our standard condition in the presence of 10 µM [3H]tetracycline and 50 µM CoCl2. Under this condition, the initial rate of the active uptake of tetracycline by the wild-type vesicles was 2.7 nmol of tetracycline per milligram of membrane protein per 30 s. The inverted vesicles prepared from the Ala70 and Ser70 mutant cells retained less than 4% of the wild-type transport activity (
